Cloning and characterization of heat shock protein gene(s) from Aegilops speltoides (Tausch) Gren. and their association with heat tolerance
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Date
2015
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PAU
Abstract
Wheat is staple food for half of the world population. Wheat yields are increasing at 0.9% annually, which is much less than the requisite increase to meet its demand in 2050. Wheat production is often limited by continual or terminal heat stress and not much is known about the mechanisms conferring olerance to heat stress. Heat shock proteins are known to play an essential role in preventing deleterious effects of high temperature and in many plant species HSP101 has a central role in heat stress survival. Aegilops speltoides, a close relative of B genome of bread wheat has been observed to confer tolerance to terminal heat stress. The present study, therefore, aimed at characterization of heat shock protein gene HSP101 in Ae. speltoides and comparing it with other species. The Ae. speltoides and other wild and cultivated wheat genotypes were analyzed for chlorophyll content at various growth stages until maturity. Ae .speltoides, per se, had significantly higher chlorophyll content at all the growth stages, even when the temperature was above 35°C. Coding sequences of HSP101C of T. aestivum were used to design the primers for studying expression of HSP101 at varying day/night temperature regimes. Expression analysis of HSP101C gene through Quantitative RT-PCR revealed differences in their induction in wild and cultivated wheat genotypes. Two Aegilops speltoides accessions pau3583 and pau3809 showed high level of expression of HSP101C gene at higher temperature compared to bread wheat, suggesting that it might be playing a role in conferring heat tolerance. Coding sequence of HSP101C gene of T. aestivum was used to identify the whole gene sequence in T. durum and Ae.speltoides genome databases. Overlapping primers were designed to amplify the whole gene from Ae. speltoides, Ae. tauschii, T. monococcum, T. durum and T. aestivum. Amplification was successful for all the fragments in all the species, however, clean sequence could be obtained in only one accession of Ae. speltoides acc pau3583. The HSP101C gene of Ae. speltoides acc. pau3583, designated as AsHsp101Cpau3583 is 4133 bp long with 2667 bp of coding sequence encoding an ORF of 888 amino acids. The AsHSP101C-pau3583 gene sequence contains more than 50 SNPs compared to AsHSP101C-TGAC. In silico comparative analysis of sequence of HSP101C of T. aestivum, Ae. speltoides, Ae. speltoides acc. pau3583, T. durum cv cappelli, T. durum cv strongfield, T. monococcum, Ae. tauschii and T. urartu HSP101C protein showed that multiple conserved domains (AAA, AAA+2, ClpB, ClpN, ClpD domains) are present. All ClpB/HSP100 genes in wheat share conserved nucleotide-binding domains. There appears to be HSP101C protein (encoded by Aegilops speltoides pau3583) that are variably homologous to proteins encoded by above wheat species throughout the entire amino acid sequence. The above eight wheat species Hsp101C gene show significant similarities in the signature sequences known to be conserved among Hsp100 proteins. The protein models of HSP101C in all eight wheat species provides high information for the ATP-binding motifs within the nucleotide binding domains (NBD) which are specific for the chaperone activity and knowledge about the mutagenic sites. These findings are important for further dissection of the molecular mechanisms underlying the stress response and for understanding the functions of the HSP100 fami ly members. The sequence information could also be used designing markers for precise transfer AsHSP101C-pau3583 gene into hexaploid wheat and test its role in heat tolerance.
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Keywords
proteins, wheats, genes, tolerance, planting, biological phenomena, chlorophylls, cloning, grain, heat shock proteins